Cloning and Functional Characterization of a ... - Semantic Scholar

The Journal of Neuroscience,

Cloning and Functional Characterization Receptor from Drosophila melanogaster Guoping Feng,l Frances Hannan, I,* Vincenzina Peter D. Evans,* and Linda M. Hall’

June 15, 1996, 16(12):3925-3933

of a Novel Dopamine

Reale,* Yuen Yi Hon,l Christopher

T. Kousky,’

1Department of Biochemical Pharmacology, State University of New York at Buffalo, Buffalo, New York 14260- 7200, and *The Babraham Institute Laboratory of Molecular Si,qnallin.q, Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom -

A cDNA clone is described that encodes a novel G-proteincoupled dopamine receptor (DopR99B) expressed in Drosophila heads. The DopR99B receptor maps to 99B3-5, close to the position of the octopamine/tyramine receptor gene at 99Al OBl, suggesting that the two may be related through a gene duplication. Agonist stimulation of DopR99B receptors expressed in Xenopus oocytes increased intracellular Ca*+ levels monitored as changes in an endogenous inward Ca*’ dependent chloride current. In addition to initiating this intracellular Ca’+ signal, stimulation of DopR99B increased CAMP levels. The rank order of potency of agonists in stimulating the chloride current is: dopamine > norepinephrine > epinephrine > tyramine. Octopamine and Shydroxytryptamine are not active (cl00 PM). This pharmacological profile plus the second-

messenger coupling pattern suggest that the DopR99B receptor is a Dl -like dopamine receptor. However, the hydrophobic core region of the DopR99B receptor shows almost equal amino acid sequence identity (40-48%) with vertebrate serotonergic, ~rl- and P-adrenergic, and Dl -like and D2-like dopaminergic receptors. Thus, this Drosophila receptor defines a novel structural class of dopamine receptors. Because DopR99B is the second dopamine receptor cloned from Drosophila, this work establishes dopamine receptor diversity in a system amenable to genetic dissection.

Dopamine is a neurotransmitter in both the central and the peripheral nervous systems and is involved in a variety of important physiological and behavioral processes, such as neuroendocrine function, emotion, and motor control (Civelli et al., 1993). Moreover, dopaminergic systems have been implicated in several neurological disorders, including Parkinson’s disease and schizophrenia (Seeman et al., 1984; Goldstein and Deutch, 1992; Wichmann and DeLong, 1993). Dopamine exerts its effects by interacting with G-protein-coupled, heptahelical membrane receptors on the cell surface (Jackson and Westlind-Danielsson, 1994). Numerous agonists and antagonists of dopamine receptors have been discovered in the search for new drugs to battle neurological disorders. Using these drugs, dopamine receptors were originally

classified into Dl and D2 subtypes based on their pharmacological specificity with neuroleptics (Kebabian and Came, 1979) a class of dopamine antagonists used to alleviate the main symptoms of schizophrenia (Levinson, 1991). Molecular cloning of dopamine receptors has revealed at least five distinct subtypes (Dl-D5) that can be divided into two classes, the Dl-like (Dl and D5) and DZlike (D2, D3, and D4) receptors based on their sequence similarity and pharmacological profile (Gingrich and Caron, 1993). These two classes of dopamine receptors are linked to distinct cascades for signal transduction. Activation of Dl-like receptors stimulates adenylyl cyclase and phosphatidylinositol4,5-bisphosphate (PI) metabolism, whereas D2-like receptor activation inhibits adenylyl cyclase and activates potassium channels (Gingrich and Caron, 1993; Jackson and Westlind-Danielsson, 1994). Dopamine and dopamine receptors have long been thought to play an important role in the invertebrate nervous system (Weiss and Drummond, 1981; Sonetti et al., 1987; Walker and HoldenDye, 1989; Ali and Orchard, 1994; Hall, 1994). We report here the molecular cloning and functional characterization of a novel Drosophila dopamine receptor that shows a pharmacology and second-messenger coupling pattern similar to Dl-like receptors. However, this Drosophila receptor differs structurally from the Dl-like group of dopamine receptors that includes mammalian Dl and D5 subtypes, and it also differs from a previously reported Dl-like receptor from Drosophila (Gotzes et al., 1994; Sugamori et al., 1995). Northern blot analysis suggests that this novel Drosophila receptor is expressed in both central and peripheral nervous systems. Stimulation of these receptors, expressed in Xenopus oocytes, generates a calcium signal and increases CAMP

Received Nov. 30, 1995; revised March 26, 1996; accepted April 2, 1996. This work was supported by National Institutes of Health Jacob Javits Neuroscience Investieator Award NS16204 to L.M.H. and NATO Collaborative Research Grant 900709 70 P.D.E. and L.M.H. G.F. was supported, in part, by a predoctoral fellowship from the Pharmaceutical Manufacturers Association Foundation. V.R. was suppbrted, in part, by a grant from the Isaac Newton Trust. C.T.K. was supported by National Institutes of Health training Grant GM0714.5 and by a Howard Hughes Predoctoral Fellowship. We thank Jane Pusey-Lee for outstanding technical assistance in the initial stages of cloning and sequencing the 0.68 PCR fragment, and Dr. Todd R. Jackman for helpful comments. Correspondence should be addressed to Linda M. Hall, Department of Biochemical Pharmacology, State University of New York at Buffalo, 329 Hochstetter Hall, North Campus, Buffalo, NY 14260-1200 (please note that Dr. Hall is on sabbatical until Y/30/96. Her sabbatical address should be used for correspondence concerning this manuscriot during that time). Dr. Hall’s sabbaticz address (9/95 to 9/30/96): Department of Microbiology and Molecular Genetics, Medical Sciences I Bldg C, Room 266, University of CaliforniaIrvine, Irvine, CA 92717-4025. Dr. Feng’s current address: Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. Copyright 0 1996 Society for Neuroscience 0270-6474/96/163925-09$05.00/O

Key words: cloned dopamine receptor; Drosophila melanogaster; Xenopus oocyte expression; adenylyl cyclase; calcium; gene mapping

Feng et al. Drosophila Dopamine Receptor

3926 J. Neurosci., June 15, 1996, 76(12):3925-2933

levels. Gene mapping provides a first step toward future studies aimed at mutant dissection of the physiological roles of dopamine receptor subtypes in the intact organism, including their possible involvement in memory and learning.

MATERIALS AND METHODS PCR amplification for initial isolation qf new receptor genomic DNA. The forward primer (OPS3: CATAGCCCTCGACCGGTACT) encodes the cytoplasmic end of the third transmembrane domain of the Drosophila octopamineityramine receptor (OctyR99AB) (Arakawa et al., 1990). The reverse primer (OPS4: GGCAGCCAGCAGATGACGAA) encodes the middle region of transmembrane domain VI from this receptor. The PCR template was 300 ng of Drosophila genomic DNA prepared from wildtype ~_ Canton-S adult flies (Jowett, 1986). The 100 ~1 PCR mixture contained 0.2 mM each of‘deoxyribonucleotide triphosphate, 10 tIIM Tris-HCI buffer. DH 8.3. 50 IIIM KCI. 1.5 mM M&l,. 0.001% gelatin. 0.1 pM of each primer, and i.5 units of AmpliTaq DkA’polymerage (PeikinElmer, Norwalk, CT). PCR was performed under reduced stringency annealing conditions: 94°C for 2 min, 33°C for 2 min, 72°C for 2 min for 6 cycles followed by an additional 31 cycles with an annealing temperature of 42°C instead of 33°C. Final extension was 10 min at 72°C. PCR products were analyzed by electrophoresis of 10 ~1 of reaction mixture on a 1% agarose gel. DNA sequencing Direct sequencing of the PCR product was pcrformcd as described p&&sly (Fenict al., i995; Zheng it al., 1995) using a Tuq Dve Primer Cvcle Seaucncine kit (Applied Biosvstems, Foster City, CA). TG facilitate (he scqiencing-of th‘e LbNA clone, nested deletions weic used (Henikoff, 1987). Each segment of DNA was scqucnccd at lcast twice in both directions. The contig was assembled using Geneworks software (Intelligcnetics, Mountain View, CA). Screeningfor cDNA clones. The 0.68 kb fragment from the initial PCR amplification experiment was labeled with [ol-“P]dCTP (-110 TBqi mmol) using the Multiprimc DNA labeling system (Amersham, Arlington Heights, IL) and was used to screen a Drosophila head cDNA library (Itoh et al., 1985) in hgtll, generously provided by Dr. Paul Salvaterra (Beckman Research Institute, Duarte, CA). Four positive clones were identified by high-stringency (Sambrook et al., 1989) screening of 4 X lo” plaque-forming units (pfu$ ‘?he longest insert (4 kb) was cut out with EcoRI and subcloned into pBluescript II SK( -) for further analysis. This insert and the gene that encodes it are referred to as DopR99B throughout this paper. Northern blots. Heads, bodies, and appendages (legs and antennae) were isolated from frozen adult flies (Schmidt-Nielsen et al., 1977). Poly(A)+ RNA and blots were prepared as described previously (Feng et al., 1995; Zheng et al., 1995). Poly(A)+ RNA (10 &lane) was run on a denaturing, formaldehyde agarose (0.8%) gel. Blots were probed with the 3’P-labeled 0.68 kb PCR fragment added to a final concentration of 10h cpmiml. After high-stringency washing (Feng et al., 1995; Zheng et al., 1993 blots were exposed to x-ray film for 24 hr at -70°C. In situ hybridization to salivary gland chromosome squashes. The 0.68 kb PCR fragment was biotinylated, hybridized to larval salivary gland polytene chromosome squashes, and localized using the Gibco (Gaithersburg, MD) Bluegene detection kit as described by Engels et al. (1985) with minor modifications (Feng et al., 1995; Zheng et al., 1995). Expression in Xenopus oocytes. Sense cRNA was prepared in vitro from the DopR99B clone in the pBluescript II SK(-) vector using T7 RNA polymerase (Stratagene, La Jolla, CA) after linearizing the plasmid with Not1 (Promega, Madison, WI). Transcripts were capped by adding 0.75 units of m7G(5’)ppp(5’)G (Boehringer Mannheim, Indianapolis, IN) to a standard 150 ~1 transcription reaction (Stratagene kit). Stage V and VI oocytes from virgin female adult Xenopus luevis were manually separated and placed in sterile ND96 medium [in mM: NaCl 96, KC1 2, CaCI, 1.8, MgCI, 1, HEPES buffer (pH 7.6) 5, containing 2.4 mM sodium pyruvate, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.2 mg/ml gentamycin]. The oocytes were defolliculated enzymatically by incubation in ND96 containing collagenase (2 mgiml) for 30 min. Oocytes were then injected with 50 ng of Drosophila DopR99B receptor sense cRNA and incubated at 19°C for 2-5 d. Uninjected oocytes were used as controls. Electrophysiological recordings were made from oocytes using a twomicroelectrode voltage-clamp technique, at a -60 mV holding potential, to measure oocyte currents (Van Renterghem et al., 1987). Oocytes were continuously superfused with ND96 during the experiments at room temperature, and drugs were added to the superfusate. CAMP assays. To monitor CAMP levels, individual oocytes were prein,

1

l

cubated for 30 min in ND96 plus 100 pM isobutylmethylxanthine (IBMX). Experimental oocytes were incubated for a further 30 min with the desired concentration of agonist in the same medium while control oocytes (to measure basal CAMP levels) were incubated in parallel in the same medium without agonist. After the incubations, each oocyte was homogenized in 500 ~1 of acidified ethanol, centrifuged to remove particulate matter, and the supernatant was evaporated to dryness in a vacuum centrifuge (Savant, Farmingdale, NY). Each sample was taken up in 60 ~1 of assay buffer and assayed for CAMP using a commercial assay kit (Amersham). Drugs. The drugs used in the classification of the expressed receptor were obtained from the following sources: dopamine hydrochloride, (-)-norepinephrine hydrochloride, (-)-epinephrine, tyramine DL-octopamine hydrochloride, 5-hydroxytryptamine hydrochloride, hydrochloride, (?)-isoproterenol hydrochloride, phentolamine hydrochloride, and m-propranolol were from Sigma (Poole, Dorset, UK); R(+)-SKF-38393 [R(+)-1-phenyl-2,3,4,5-tetrahydro-(lH)-3benzazepine-7,8-dial], quineloranedihydrochloride, (-)-quinpirole hydrochloride, PD-128,907 [(+)-(4aR,lObR)-3,4,4a,lOb-tetrahydro4-propyl-2H,SH-(l)benzopyrano-(4,3-b)-l,4-oxazin-9-ol-hydrochloride], cis-(Z)-flupenthixol dihydrochloride, R( +)SCH-23390 [R(+)-7chloro-8-hydroxy-3-methyl-l-phenyl-2,3,4,5-tetrahydro-IH-3-bcnzepine hydrochloride], S( -)-sulpiride, spiperone hydrochloride, (+)butaclamol hydrochloride, S(-)-cticlopridc hydrochloride, domperidone, (+)-bromocriptine methanesulfonate, (+)-h-chloro-APB [( ?) -6-chloro-7,8-dihydroxy -3-allylI -phenyl-2,3,4,5-tctrahydroI H-3R(+)-6-bromo-APB (R( +)-&bromobenzazepinc hydrobromidcl, 7, 8-digydroxy:3ally1 - I- phenyl -i,3;4,5- tetrahydro- I G-T&enzazepine hvdro-bromidcj. I t-)-6-chloro-PB I( + 1-6-chloro-7&dihvdroxvlpienyl-2,3,4,5-fctrah‘yd& I H-3-benzazepine hydrobroniide], :,lnd CL)PPHT [(~)-2-(N-phenylethyI-N-propyl)amino-5-hydroxytctr~~lin hydrochloride] wcrc from Rcscarch Biochcmicals (Natick, MA).

RESULTS Cloning of a novel Drosophila When

we

began

these

studies,

dopamine cDNAs

receptor encoding

several

G-protein-coupled receptors had been cloned from Drosophila, including a muscarinic acetylcholine receptor (Onai et al., 1989) several serotonergic receptors (Witz et al., 1990; Saudou et al., 1992), and an adrenergic receptor homolog (OctyR99AB) (Arakawa et al., 1990). However, there were no representatives of the dopaminergic receptor family cloned from invertebrates even though the role of dopamine was well established in the invertebrate nervous system. To expand the group of cloned G-protein-coupled receptors from Drosophila, we used reduced stringency PCR with primers from regions conserved in all biogenic amine receptors. To facilitate the identification of those PCR products that were likely to encode new receptors, we amplified across a region including cytoplasmic loop 3 (between transmembrane domains V and VI). This nonconserved loop varies greatly in length among different G-protein-coupled receptors (Probst et al., 1992), so different size amplification products are expected and would serve to distinguish fragments encoding new receptors from those encoding previously

cloned

ones.

In addition,

the amplified

region

also

includes the conserved transmembrane domains IV, V, and part of VI. Because these domains are easily recognized, this allows rapid identification of these amplified fragments encoding G-protein-coupled receptors. PCR primers were chosen by aligning the amino acid sequence of the Drosophila octopamine/tyramine receptor (OctyR99AB) (Arakawa et al., 1990) with sequences of a subset of previously cloned vertebrate biogenic amine receptors. A forward primer sequence (OPS3) was selected from the region of the Drosophila octopamine/tyramine receptor cDNA that encodes the cytoplasmic end of the third transmembrane domain (amino acids IALDRYW). This included the DRY sequence that is found in almost

Feng et al. .

DrosophilaDopamine

J. Neurosci.,

Receptor

June 15, 1996, 16(12):3925-2933

3927

A CCGAGCAGCAACTGCCGGGGAAATTGGAACAGGAACATAACTCGCGTCAGCTGCAGCGA

CAGCACTGAGTTGCAATGGTGGACGACA * M " D D

120 F

5 240 45

GTTACAACATCTCCGAGGATGTCTACTTCTACTTC~TGGGCTGCCCACGAGCACGGAGCTCGTGCTG~CGCCACCACCTCCGCCACCAGCGCCACCTTGAGTCCTGCGATGGTGGC~ YNISEDVYFYFNGLPTSTELVL;ATTSATSATLSPAMVAT l

360 85

CAGGAGGTGGCGGCACCACCACGCCGGAACCCCGATCTCTCCGAGTTCCTGGAGGCGCTGCCC~CGACCGTGTGGGCCTGCTGGCCTTCCTCTTCCTGTTCTCCTTCGCTACGGTTTTCG L E A L PNDRVG GGGGTTTPEPDLSEF LLAFLFLF SFATVFG

480 125

I 600 165

GCAACTCACTGGTCATCCTCGCCGTCATCCGGGAGCGGTACTTGCACACGGCCACC~CTACTTCATCACCAGCCTGGCCGTGGCCGACTGCCTCGTGGGCCTGGTGGTCATGCCCTTCT NSLVILAVIRERYLHTATNYFI TSLAVADCLVGLVVMPFS

II 720 205

CAGCGCTCTACGAGGTGCTGGAGAACACGTGGTTCTTCGTGGTTCTTCGGCACGGACTGGTGCGACATCTGGCGGTCCCTGGACGTGCTCTTCAGCACAGCCTCCATACTG~TCTGTGCGTGATCTCAC ALYEVLENTWFFGTDWCDIWRSLDVLFSTASILNLCVISL

III 840 245

TCGACCGCTACTGGGCCATCACGGATCCCTTTAGCTATCCCATGAGGATGACGGTC~GCGAGCGGCCGGTCTGATAGCCGCTGTATGGATCTGCTCCAGCGCCATTAGCTTTCCGGCCA ICSSAISFPAI DRYWAITD PFSYPMRMTVKRAAGLIAAVW

A

Iv

TTGTGTGGTGGCGAGCGGCGAGGGATGGCGAGATGCCCGCCTAC~GTGCACCTTCACCGAGCACCTGGGCTACCTAGTCTTCTCGTCGACGATATCCTTCTACCTGCCGCTTCTAGTGA VWWRAARDG EMPAYKCTFTEHLGYLVFSSTISFYLP V TGGTCTTCACCTACTGTCGCATCTACAGGGCAGCCGTCATCCAGACGAGATCTCTT~GATTGG~CC~GCAGGTGCTCATGGCCTCCGGGG~CTGCAGCTCACATTGCGTATTCATC VFTYCRIYRAAVIQTRSLKIGTKQVLMASGELQLTLR

A

A

960 285

LLVM

I

H

R

1080 325 1200 365

GTGGTGGCACTACGCGGGATCAGC-CCAGGTCTCCGGAGGAGGAGGTGGTGGAGGAGGAGGTGGCGGTGGCGGAGGATCTCTGAGCCACTCGCACTCCCATTCGCACCATCATCATC GGTTRDQQNQVSGGGGGGGGGGGGGGSLSHSHSHSHHHHH

A ACAATCACGGTGGTGGCACGACGACCTCCACGCCGGAGGAGCCGGATGATGAGCCGCTATCCGCTCTGCAT~C~CGGACTGGCACGCCATCGGCACATGGGC~G~CTTCTCGCTGT SALHNNGLARHRHMGKNFSLS NHGGGTTTSTPEEPDDEPL

A

1440 445

CCAGGAAACTGGCGAAGTTCGCCAAGGAGAG~G~GGCGGCC~GACGCTGGGCATCGTGATGGGCGTGTTCATCATCTGCTGGCTGCCCTTCTTCGTGGTC~CCTGCTGTCCGGGTTCT RKLAKFAKEKKAAKTLGIVMGVFIICWLPFFVVNLLSGFC VI GCATCGAGTGCATCGAGCACGAGGAGATCGTCTCGGC~TCGTCACCTGGCTCGGCTGGATC~CTCCTGCATG~TCCTGTGATTTACGCCTGCTGGAGCAGGGACTTTCGCAGGGCCT IECIEHEEIVSAIVTWLGWI NSCMNPVIYACWSRDFRRAF VII TTGTGCGTCTGCTGTGCATGTGCTGTCCACGC~GATTCGCCGC~GTACCAGCCCACGATGCGTTCC~GTCGCAGAGATTCGCGACGCGGCGCTGCTACTCGACCTGCTCGCTGCACG VRLLCMCCPRKIRRKYQPTMRSKSQRFATRRCYSTCSLHG

A

A

1560 485 1680 525

A

GCATTCAGCACGTGCGACACAACTCCTCCTGCGAGCAGACCTACATATAGTTTAGCGTAGATTTAGTTGAC~TGATT~GGCGCCGAGCCACGCCCCCTAGCGCCCTCCATTTGTCCGT~G l IQHVRHNSCEQTYI'

1320 405

l

1800 539 1920 2040 2160 2280 2400 2520 2640 2760 2880 3000 3120 3240 3360 3480 3600 3720 3840 3960 4003

B 400 200 0 -200 I 100

I 200

I 300

I 400

I 500

Amino acid position Figure 1. The DopR99B sequence.A, Nucleotide sequence and deduced amino acid sequence of the Drosophila dopamine receptor DopR99B. In-frame stop codons are indicated by asterisks. The sequence of a potential ribosome binding site is boxed. The seven transmembrane domains are underlined and numbered. Potential PKC phosphorylation sites are indicated by A. Potential N-glycosylation sites are marked by + B, Hydrophobic@ plot of the deduced DopR99B amino acid sequence. Regions above the line are hydrophobic. Transmembrane domains are numbered. The hydropathy plot was done using the method of Kyte and Doolittle (1982) and the Geneworks software (Intelligenetics). The Genbank accession number for DopR99B is U34383.

3928 J. Neurosci.,

Feng et al. . Drosophila

June 15, 1996, 76(12):3925-2933

Pathway Accession #

Shtdror Shtratlb Shtratld Shthumle Shtratla OctyR99AB DoDR~~B

t PLC 1‘ PLC t PLC 1 AC LAC t AC 1 AC IAC IAC SAC I AC t AC : AC 1 AC 1 AC

M21410 M30705 X66842 211489 z11490 M55533 X62944 M89953 M91467 JO5276 M60789 U34383 M6237 1 M62372

:AC 7 AC t AC 7AC 7 AC ?

!;%:, M36831 x59500 x53944 X58497 X55760 M35077 X58454 M69118 X77234 M60654

i AC 7AC I‘AC

J”o::::” JO5561 M74716

:AC

Figure 2. Structural relationship of DopR99B with other G-proteincoupled receptors. Sequence alignment was done with the PILEUP program of the Wisconsin Genetic Computer Group (GCG) software (Devereux et al., 1984) using the hydrophobic cores of each receptor listed. The hydrophobic core was defined as the region between and including transmembrane domains I and V plus the region between and including transmembrane domains VI and VII. The lengths of the horizontal lines are inversely proportional to the percentages of sequence similarity between receptors or groups of receptors. In the receptor names listed, the first three to four letters refer to the general receptor type: Adr, adrenergic; Dop, dopamine; %t, serotonin; Octy, octopamineityramine. The next letters in the name refer to the species: duo, Drosophila; hum, human; rat, rat; xen, Xenopus. The exceptions to this nomenclature are three Drosophila receptors (DopRSEF, DopRYYB, OctyR99AB). For these, the type name is followed by R (receptor) and then the salivary gland chromosome map position. Pathway refers to the second-messenger coupling reported in the literature for each receptor: PLC, phospholipase C; AC, adenylyl cyclase. An upward arrow indicates stimulation of the indicated secondmessenger system, and a downward arrow indicates inhibition. Accession # refers to the Genbank database locator number.

all cloned G-protein-coupled receptors. A reverse primer sequence (OPS4) was from the conserved middle region of transmembrane domain VI of OctyR99AB cDNA (encoding amino acids FVICWLP). Genomic DNA was used as the template to avoid assumptions concerning the time- and tissue-specific expression of receptors. Using the OPS3 and OPS4 PCR primers, five fragments (1.7, 1.0, 0.68, 0.60, and 0.46 kb) were amplified from Drosophila genomic DNA. Direct sequencing of the PCR products revealed that the 1.7 kb fragment is the genomic equivalent of OcfyR99AB with a 0.7 kb intron in the region encoding cytoplasmic loop 3. The 0.68 kb fragment had high sequence similarity to OctyR99AB and other G-protein-coupled receptors in the transmembrane domains, but was different in cytoplasmic loop 3. These data suggested that this 0.68 kb fragment encodes part of a novel

Dopamine

Receptor

G-protein-coupled receptor gene in Drosophila. Using this 0.68 kb PCR fragment as a probe, four overlapping cDNA clones were isolated from an adult head cDNA library. The clone with the longest insert (4 kb) was completely sequenced and has been designated DopR99B.

Structural

features

of the DopR99B cDNA sequence

The 4 kb cDNA insert contains an open reading frame encoding 538 amino acids with a predicted molecular mass of 59.5 kDa (Fig. L4). The open reading frame is defined by the first in-frame ATG that is preceded by an in-frame stop codon. Although the flanking sequence preceding this ATG (-4 to - 1, TGCA) does not match well with the Drosophila consensus sequence for a translation start site [C/A A A A/C (Cavener, 1987)], a potential ribosome-binding site around this ATG was identified (Fig. L4). A comparison of the cDNA sequence in the regions used for the original PCR primers shows that in the forward primer region there are 4 base mismatches concentrated in the first 7 nucleotides from the 5’ end. The reverse primer had only 1 base mismatch in the fourth base position from the 3’ end. The robust amplification of the 0.68 kb product indicates that these mismatches were all tolerated at the initial annealing temperature of 33°C.

Sequence comparison with other G-protein-coupled receptors The deduced amino acid sequence for DopR99B shows many standard characteristics of the G-protein-coupled receptor gene family. The hydropathy plot (Fig. 1B) reveals the 7 transmembrane domains diagnostic of all members of this group of receptors (Probst et al., 1992). At the cytoplasmic end of transmembrane domain III is the highly conserved DRY sequence. This amino acid triplet is thought to be important in G-protein coupling (Dixon et al., 1987; Fraser et al., 1988). Single cysteine residues (at 182 and 261) in extracellular loops 1 and 2, respectively, are also conserved. These residues form a disulfide bond that stabilizes the functional receptor structure (Dixon et al., 1987; Karnik et al., 1988; Fraser, 1989). Two aspartate residues in TM II and TM III that are conserved in all catecholamine receptors are also conserved in DopR99B (D154 and D189). These two aspartates are thought to play a direct role in binding the amine groups on catecholamines (Strader et al., 1988). The three serine residues in TM V that are postulated to interact with the catecholamine ring hydroxyl groups (Strader et al., 1989; Pollock et al., 1992) are also conserved in DopR99B (S273, S274, S277). Thus, this new receptor has appropriately placed amino acid side chains for binding catecholamines. There are other, more genera1 structural motifs that are found in this Drosophila receptor as well as in other G-protein-coupled receptors. For example, mammalian dopamine receptors typically have one to four N-linked glycosylation sites in their extracellular domains (Jackson and Westlind-Danielsson, 1994). This Drosophila receptor has four potential N-linked glycosylation sites in the amino terminus (positions 5, 31, 47, and 68). This region is likely to be an extracellular domain. All G-protein-coupled receptors have protein kinase C sites in cytoplasmic loop 3 (between TM V and VI) and in the C-terminal tail, but the exact positions of these sites are not strictly conserved. This newly cloned Drosophila receptor has eight potential protein kinase C phosphorylation sites in cytoplasmic loop 2 (position 222), cytoplasmic loop 3 (positions 302, 320, 328, and 405), and in the C terminus (positions 504, 509, and 514). Finally, many G-protein-coupled receptors undergo a post-translational palmitoylation at a conserved

Feng et al. . Drosophila

Dopatmne

Receptor

cysteine residue in the C-terminal tail (Gingrich and Caron, 1993). This modification serves to anchor the tail to the internal face of the plasma membrane, creating a fourth cytoplasmic loop. The Drosophila receptor has several cysteines that could serve this function (at 490, 492, 493, 517, 521, and 534). The conserved structural motifs, along with the fact that this gene product has, overail, a high sequence similarity with other G-protein-coupled receptors, suggest that it encodes a novel G-protein-coupled receptor in Drosophila. Comparison of the predicted hydrophobic core region of the DopR99B deduced amino acid sequence revealed significant sequence similarity with vertebrate and invertebrate dopamine receptors (41-47% identity), serotonin receptors (40-47% identity), cul- and /3-adrenergic receptors (46-48% identity), as well as the Drosophila octopamineityramine receptor from which the primers were derived (47% identity). Detailed sequence comparison of either the hydrophobic core region or the full-length deduced amino acid sequence showed that the DopR99B receptor clearly belonged to the biogenic amine class of receptors, but it was not possible to predict its receptor type based on sequence comparisons alone. The dendrogram in Figure 2 shows that DopR99B did not fall clearly within one of the biogenic amine receptor groups, although a previously described (Gotzes et al., 1994; Sugamori et al., 1995) Drosophila dopamine receptor (DopR35EF) consistently grouped with the vertebrate Dl-like dopamine receptors. To define the DopR99B receptor-type pharmacologically, we expressed it in Xenopus oocytes. Expression studies in Xenopus oocytes Xenopus oocytes translate exogenous mRNAs encoding neurotransmitter receptors and incorporate the receptor proteins into their cell membranes (Sumikawa et al., 1981; Barnard et al., 1982). To define which ligands activate the DopR99B receptor, we expressed its cRNA in Xenopus oocytes and tested the ability of a range of biogenic amines to initiate responses. The application of dopamine initiated transient inward current responses in injected oocytes (Fig. 3A). Uninjected, control oocytes showed no responses (data not shown). The responses have a reversal potential of -21.7 5 2.9 mV (n = 3) estimated from current-voltage (1-v) plots, consistent with their mediation via the activation of the endogenous, inward calcium-dependent chloride current of the oocyte. This inward current is presumably generated by the same mechanism originally reported by Masu et al. (1987) after stimulation of another G-protein-coupled receptor (the bovine substance-K receptor) expressed in oocytes; i.e., receptormediated activation of phospholipase C increases PI hydrolysis and stimulates release of intracellular calcium which, in turn, activates the endogenous calcium-dependent chloride current of the oocyte. Dose-response curves (Fig. 3B) indicate that the threshold response to dopamine occurred in the region of 1 nM, whereas the maximal response occurred at 1 pM. Exposure of injected oocytes to concentrations of dopamine >l pM consistently produced smaller responses (Fig. 3AJ). Figure 3B also shows that the catecholamines norepinephrine and epinephrine induce inward currents in injected oocytes. However, these catecholamines are less effective than dopamine. The injected oocytes also showed inward currents when exposed to tyramine (Fig. 3B), but much higher concentrations were required and the average current response was smaller at the maximum concentration tested (100 PM) than for the other amines. Oocytes showed no response to either oL-octopamine or 5hydroxytryptamine at concentrations

J. Neurosci.,

June 15, 1996, 16(12):3925-2933

3929

A 1nM

1OOnM

10nM

1 , .. . .r

lOOnA 100s

B

200

I

+ -A+

(-)-Norepinephrine (-)-Epinephrine Tyramine

t I

-10

I

-9

-8

-7

I

-6

T

I

-5

-4

Log [Agonist] (M) Figure 3. Inward current response after agonist stimulation mediated by the DopR99B receptor expressed in Xenopus oocytes. Two minute pulses of agonists were given to Xenopus oocytes 3 d after the injection

of

DopR99B cRNA. A, Typical responses of a single oocyte to various concentrations of dopamine. B, Dose-response curves for dopamine, (-)-norepinephrine, (p)-epinephrine, and tyramine. The results (from at least 6 oocytes) are expressed as the mean peak inward current t SE initiated by each amine.

up to 100 pM. Taken together, these results suggest that the DopR99B cDNA clone encodes a Drosophila dopamine receptor. To determine whether this Drosophila dopamine receptor falls into the Dl-like or D2-like pharmacological category, the ability of antagonists (10 PM) to block the responses to 1 pM dopamine

3930 J. Neurosci.,

Feng et al.

June 15, 1996, 76(12):3925-2933

l

DrosophilaDopamine

Receptor

Table 1. Effects of agonists and antagonists on inward currents in Xenopus oocytes expressing the Drosophila DopR99B receptor --O--

A Antagonists (I 0 FM)

% of Response to 1 PM dopamine

Flupenthixol R (+) SCH23390 S (p)-sulpiride Spiperone (+jButaclamol Phentolamine m-Propranolol S( p)-eticlopride Domperidone

3.9 2 34.2 k 56.8 2 63.0 t 66.7 t 11.6 t 89.6 k 103.2 ? 118.3 5

Receptor-type specificity (?I = (n = (n = (n = (n = (n = (n = (n = (n =

1.6% 8.9% 16.1% 9.2% 4.5% 7.1% 5.9% 10.9% 7.0%

cRNA injected Control

5) 6) 5) 7) 6) 6) 7) 4) 3)

Dl/D2-like Dl/D5 D2-like D2-like DUDl-like ol-Adrenergic p-Adrenergic D2-like Peripheral D2

B Agonists (10 FM)

% of Response to 1 FM dopamine

(?)-h-Chloro-APB R (r)-6-bromo-APB (t)-6-Chloro-PB Quinelorane ( z)-Bromocriptine Quinpirole R (+)-SKF-38393 (*)-Isoproterenol PD-128,907 (?)-PPHT

89.4 c 3.8% 73.3 k 9.1% 19.6 t 7.1% 11.3 * 3.0% 7.5 k 2.6% 4.1 z 2.3% 1.8 * 1.2% 1.3 2 0.9% 0 0

Receptor-type specificity (n (n (n (n (n (n (n (n (n (n

= = = = = = = = = =

6) 6) 5) 8) 5) 6) 6) 6) 6) 4)

Dl-like Dl-like Dl -like D2-like D2-like D2iD3 Dl-like /3-Adrenergic D3 D2-like

Inward currents were initiated by 2 min pulses of 1 ELM dopamine. The mean response to a 2 min control pulse of 1 ELMdopamine was 331.4 -C 15.6 nA (n = 73). A, The size of the response to a dopamine pulse given in the presence of 10 /UVI antagonist is expressed as a percentage ? SE of the response to a control dopamine pulse given to the same oocyte. Antagonists alone did not initiate any currents. 8, Agonists were applied as 2 min pulses. The inward currents generated are expressed as the percentage i- SE of the response of the same oocyte to a control dopamine pulse.

in oocytes expressing the DopR99B receptor was determined (Table 1A). The rank order potency of the dopaminergic antagonists tested was: flupenthixol > R( +)-SCH-23390 > S(-)sulpiride > spiperone > (+)butaclamol > S( -)eticlopride = domperidone. The Dl-like receptor blockers were, in general, Table 2. Effects of biogenic amines on CAMP levels in Xenopus oocytes expressing the Drosophila DopR99B receptor

Agonist

DopR99B cRNA-injected

Basal Dopamine (-)-Epinephrine (p)-Norepincphrine Phenylethylamine 5.Hydroxytryptamine DL-Octopaminc Phenylethanolamine Tyramine

0.82 2 0.05 1.46 + 0.10 1.28 2 0.15 1.15 ? 0.06 0.86 i 0.09 0.83 ? 0.09 0.78 +- 0.07 0.76 2 0.06 0.71 ? 0.08

Uninjected (n (n (n (n (n (n (n (n (n

= = = = = = = = =

9) 10) 8) 8) 10) 5) 10) 10) 10)

0.87 + 0.08 0.77 i 0.09 0.76 + 0.08 0.71 2 0.07 0.69 2 0.10 0.96 5 0.05 0.69 i 0.08 0.89 !z 0.06 0.71 f 0.09

(n (n (n (n (n (n (n (n (n

= = = = = = = = =

10) 10) 10) 9) 10) 5) 10) 10) 9)

CAMP levels were measured 5 d after cRNA injection. Before measurement, oocytes were preincubated for 30 min in 100 WM IBMX and then exposed to 10 ELMbiogenic amine for 30 min in the presence of 100 +VI IBMX. The results are expressed as the mean oocyte CAMP level (pmolioocyte) 2 SE.

I

I

I

I

B

-7

-6

-5

-4

Log [Dopamine] (M) Figure 4. Dose-response curves for increase in CAMP levels in oocytes expressing DopR99B. Five days after injection with Drosop/zila DopR99B receptor cRNA, injected and uninjected (control) oocytes were treated with the indicated dopamine concentrations for 30 min in the presence of 100 PM IBMX after preincubation for 30 min in 100 PM IBMX. The results are expressed as the mean oocyte CAMP level (pmol/oocyte) 2 SE (n = 5 oocytes). B, Basal levels. more effective than those with D2-like receptor specificity. The oc-adrenergic blocker phentolamine and the P-adrenergic blocker DL-propranolol were poor blockers, falling within the same range as the weaker D2-like receptor blockers. Thus, in terms of antagonist responses, the DopR99B receptor would be classed with the Dl-like dopamine receptor group. When a range of synthetic dopamine agonists specific for various vertebrate receptor subtypes was tested (Table lB), both Dl-like and D2-like agonists mimicked the dopamine responses in oocytes expressing the DopR99B receptor. However, with the exception of the relatively ineffective Dl-like agonist SKF-38393, the Dl-like agonists were more effective than the D2-like agonists. Thus, these pharmacological studies of the inward currents generated by dopamine application indicate that the receptor encoded by the DopR99B cDNA, again, has a pharmacological profile closer to Dl-like dopamine receptors. Another way to categorize receptors is in terms of the secondmessenger systems to which they couple. Therefore, we have also investigated second-messenger coupling for the Drosophila DopR99B receptor. This receptor, when expressed in Xenopus oocytes, appears to couple to multiple second-messenger systems. As described above, receptor activation initiates a calcium response. In addition, dopamine activation of the DopR99B receptor also increases CAMP levels in oocytes (Fig. 4). This response is mimicked by the application of epinephrine and norepinephrine to expressing oocytes, but not by phenylethylamine, 5hydroxytryptamine, DLoctopamine, phenylethanolamine, or tyramine when these agonists were tested at a concentration of 10 PM (Table 2). Stimulation of CAMP levels is characteristic of second-messenger coupling by Dllike receptors, whereas inhibition of CAMP synthesis is characteristic of D2-like receptors (see Fig. 2). Therefore, in this respect,

Feng et al.

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Receptor

H

J. Neurosci.,

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LJA

Figure 5. Northern blot analysis of DopR99B. A Northern blot of

poly(A)+ RNA isolated from heads (II), bodies (B), and legs/antennae (L/A) was probed with the 32P-labeled 0.68 kb PCR fragment. To control for mRNA recovery, the blot was reprobed with ribosomal protein cDNA (rp49), which is expressed throughout the organism (O’Connell and Robash, 1984). DopR99B also resembles Dl-like receptor DopR99B (for &amine

receptors. We have named this Receptor f?rom Drosophila mapping to 99B to distinguish it from another Drosophila Dl-like dopa-

mine receptor (which we designate as DopR35EF) described previously (Gotzes et al., 1994;Sugamori et al., 1995). mRNA distribution and chromosomal localization of DopR99B To determine the distribution of the DopR99B transcript in different body parts, we did Northern blot analysis with poly(A)+ RNA isolated from heads, bodies, and appendages (mostly legs and antennae). The blot was probed with the original 0.68 kb PCR fragment that contains the nonconserved cytoplasmic loop 3. As shown in Figure 5, a single band of 5.5 kb mRNA was detected predominantly in heads with a lighter signal in appendages. No expression was detected in bodies. This distribution is consistent with a role for these dopamine receptors in the central and peripheral nervous systems. The absence of major expression in bodies suggeststhat this receptor is not highly expressed in flight muscle. To determine where DopR99B maps in the Drosophila genome, the 0.68 kb PCR fragment was biotinylated and hybridized to Drosophila salivary gland polytene chromosomes (Fig. 6). The probe hybridized to a single location on the right arm of chromosome 3 at 99B3-5. This location is close to, but distinct from, the Drosophila octopamine/tyramine receptor gene that maps to 99AlO-Bl (Arakawa et al., 1990). The structural relationship between DopR99B and this Drosophila octopamineltyramine receptor (OctyR99AB, Fig. 2) suggeststhat these receptors may be related by a local gene duplication followed by independent evolution. DISCUSSION The present study establishes, for the first time, the presence of multiple dopamine receptor types in insects. These receptors, which we have designated as DopR35EF [cloned in previous studies (Gotzes et al., 1994; Sugamori et al., 1995)] and DopR99B (present study), are similar in that both stimulate adenylyl cyclase when activated. Although both resemble the vertebrate Dl-like

Figure 6. Chromosome localization of DopR99B. In situ hybridization to Drosophila salivary gland polytene chromosomes using the biotinylated

0.68 kb PCR fragment. The awow indicates the site of hybridization at 99B3-5 on the right arm of chromosome 3.

dopamine receptor class, there are differences. For example, vertebrate Dl-like receptors generally have long C-terminal tails ranging from 113 to 117 amino acids in length, whereas DZlike receptors have short tails in the range of 16-18 amino acids (Jackson and Westlind-Danielsson, 1994). The two Drosophila dopamine receptors have C-terminal tails that are intermediate in length (62 amino acids for DopR35EF and 64 for DopR99B). Although the two Drosophila receptors resemble each other with respect to C-terminal tail length, in overall sequence similarity DopR35EF is less similar to DopR99B (43% sequence identity in the hydrophobic core region) than it is to the vertebrate Dl-like class of dopamine receptors (46-48% identity). When dendrograms are constructed based on structural relatedness (e.g., Fig. 2), DopR35EF consistently falls into a group with vertebrate Dl-like dopamine receptors regardless of whether the entire sequence or only the hydrophobic core is used for comparison. DopR99B does not group with the Dl-like receptors, but instead shows almost equal sequence identity to a number of different biogenic amine receptor groups. This suggests that the dopamine receptor subtypes in Drosophila did not arise from each other but, rather, may represent convergent evolution from different precursors. Although the Drosophila DopR99B receptor does not fall into the Dl-like structural group, it does show pharmacological properties similar to Dl-like receptors (Jackson and WestlindDanielsson, 1994; Seeman and VanTol, 1994) when expressed in Xenopus oocytes. Agonists and antagonists with Dl-like selectivity are, in general, more effective than those with DZlike receptor selectivity. A comparison of our pharmacological data with the limited information on DopR35EF (Gotzes et al., 1994; Sugamori et al., 1995) suggeststhat these two Drosophila dopamine receptors differ in their pharmacological specificity. For example, the dopamine receptor agonist SKF-38393 is 30% as effective as dopamine on DopR35EF (Gotzes et al., 1994), but is almost inactive on DopR99B. With respect to antagonists, SCH-23390 is less effective than flupenthixol and butaclamol on DopR35EF, but is intermediate between flupenthixol and butaclamol on DopR99B. Agonist stimulation of DopR99B activates adenylyl cyclase to increase CAMP levels and also generates a calcium signal presum-

3932 J. Neurosci.,

June 15, 1996, 16(12):3925-2933

ably through stimulation of phospholipase C. Both of these responses have been reported for vertebrate Dl-like receptors (Figure 2) (Mahan et al., 1990). In contrast, vertebrate D%-like receptors usually show opposite effects on these secondmessenger systems because they inhibit CAMP synthesis (Jackson and Westlind-Danielsson, 1994) and are associated with a decline in intracellular Cazt levels. Structural features, presumably in the second and third intracellular loops and in the C terminus, must underlie the ability of this Drosophila dopamine receptor to couple to two different second-messenger systems. Because the DopR99B receptor is structurally distinct from classical vertebrate dopamine receptors, the use of chimeras and the application of in vitro mutagenesis may produce novel insights into how dopamine interacts with this receptor and how this interaction results in the stimulation of different systems. Our results leave unanswered the question of whether Drosophila has a D2-like dopamine receptor homolog. Dopamine and the enzymes involved in its synthesis are widely distributed in the insect nervous system (Klemm, 1976; Evans, 1980; Livingstone and Tempel, 1983; Brown and Nestler, 1985; Budnik and White, 1987). Previous studies demonstrating the presence of dopaminesensitive adenylyl cyclases in insect nervous tissue (Nathanson and Grccngard, 1973; Bodnaryk, 1979; Uzzan and Dudai, 1982; Orr et al., 1987) and salivary glands (House and Ginsborg, 1979; LafonCazal and Bockaert, 1984; Evans and Green, 1990a,b; Ali and Orchard, 1994), together with radioligand binding studies on cockroach brains (Notman and Downer, 1987), suggested the presence of Dl-like dopamine receptors in insects. These Dl-like receptor responses could correspond to either the DopR99B receptor described in this report or the DopR35EF receptor (Gotzes et al., 1994; Sugamori et al., 1995) or both. There are no reports yet of D2-like dopamine receptor activity in insects, but this is not a well studied area. None of the other PCR amplification products found in our search for new receptor types in Drosophila encode additional dopamine receptors, although at least one product (the 0.60 kb fragment) represents another G-protein-coupled receptor (G. Feng, R. Venard, and L. M. Hall, unpublished results). Thus, a modification of our cloning strategy will be necessary to search for additional dopamine receptor cDNAs in Drosophila. In insects, the dense innervation of the mushroom bodies by dopamine-immunoreactive neurons (Budnik and White, 1988; Schafer and Rehder, 1989: Nassel and Elkes, 1992), together with studies on behavioral mutants of Drosophila (Tempel et al., 1984; Buchner, 1991), has suggested roles for dopamine in both memory and learning, and in neuronal development. Analysis of the Drosophila mutant Ddc, which is deficient in dopa decarboxylase (therefore lacking dopamine and serotonin), reveals learning deficits (Tempel et al., 1984) and an abnormal pattern of neuronal arborization (Budnik et al., 1989) that can be partially rescued by feeding flies dopamine, but not serotonin. These studies suggest possible roles for cloned dopamine receptors from Drosophila. Our work, in conjunction with that of others (Gotzes et al., 1994; Sugamori et al., 1995) establishes that there are at least two genetically distinct dopamine receptors in Drosophila that both stimulate adenylyl cyclase when activated. The DopR99B receptor is also capable of generating a calcium signal. Are either of these receptors involved in the signal transduction role that dopamine plays in learning and memory? Because the DopR35EF receptors are preferentially expressed in the optic lobes (Gotzes et al., 1994), they are unlikely to play a role in learning and memory that involves mushroom body-mediated functions. In contrast,

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Drosophila Dopamine

Receptor

DopRYYB is good candidate for dopaminergic modulation of learning and memory because this receptor is expressed preferentially in mushroom bodies (K. Han and R. Davis, personal communication). Genetic studies aimed at identifying dopamine receptor mutations in Drosophila will be useful in defining their physiological and behavioral roles. REFERENCES Ali DW, Orchard I (1994) Characterization of dopamine and serotonin receptors on the salivary glands of the locust, Locustu migruforia. Biog Amines 10:195-212. Arakawa S, Gocayne JD, McCombie WR, Urquhart DA, Hall LM, Fraser CM, Venter JC (1990) Cloning, localization, and permanent expression of a Drosophila octopamine receptor. Neuron 4:343-354. Barnard EA, Miledi R, Sumikawa K (1982) Translation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes. Proc R Sot Lond [Biol] 2151241-246. Bodnaryk RP (1979) Identification of specific dopamine- and octopamine-sensitive adenylate cyclases in the brain of Mumesfra conjiguruta Wlk. Insect Biochem 9:155-162. Brown CS, Nestler C (1985) Catecholamines and indolalkylamines. In: Comprehensive insect physiology, biochemistry and pharmacology, Vol 11 (Kerkut GA, Gilbert LI, eds), pp 436-497. Oxford: Pergamon. Budnik V, White K (1987) Genetic dissection of dopamine and serotonin synthesis in the nervous system of Drosophila melanogaster. J Neurogenet 4:309-314. Budnik V, White K (1988) Catecholamine-containing neurons in Drosophila melunoguster distribution and development. J Comp Neurol 268:400-413. Budnik V, Wu C-F, White K (1989) Altered branching of serotonincontaining neurons in Drosophila mutants unable to synthesize serotonin and dopamine. J Neurosci 9:2866-2877. Buchner E (1991) Genes expressed in the adult brain of Drosophila and effects of their mutations on behavior: a survey of transmitter- and second messenger-related genes. J Neurogenet 7:153-192. Cavener DR (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res 15:1353-1361. Civelli 0, Bunzow JR, Grandy DK (1993) Molecular diversity of the dopamine receptors. Annu Rev Pharmacol Toxic01 33:281-307. Devereux J, Haeberli P, Smithies 0 (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387-395. Dixon RAF, Sigal IS, Candelore MR, Register RB, Scattergood W, Rands E, Strader Cc (1987) Structural features required for ligand binding to the B-adrenereic recentor. EMBO J 6:3269-3275. Engels WR, P&ton C’R, Thompson P, Eggleston WB (1985) In situ hybridization to Drosophila salivary chromosomes with biotinylated DNA probes and alkaline phosphatase. Focus 8:6-8. Evans PD (1980) Biogenic amines in the insect nervous system. Adv Insect Physiol 15:317-473. Evans AM, Green KL (1990a) The action of dopamine receptor antagonists on the secretory response of the cockroach salivary gland in vitro. Comp Biochem Physiol 97C:283-286. Evans AM, Green KL (1990b) Characterization of the dopamine receptor mediating the hyperpolarization of cockroach salivary gland acinar cells in vitro. Br J Pharmacol 101:103-108. Feng G, Desk P, Kasbekar DP, Gil DW, Hall LM (1995) Cytogenetic and molecular localization of tipE: a gene affecting sodium channels in Drosophila melunoguster. Genetics 139:1679-1688. Fraser CM (1989) Site-directed mutagenesis of P-adreneraic receptors: identification of conserved cysteine iesidues that independently affect ligand bindine and recentor activation. J Biol Chem 264:9266-9270. Fraskr CM, Ch;ng FZ, Wang CD, Venter JC (1988) Site-directed mutagenesis of human P-adrenergic receptors: substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase. Proc Nat1 Acad Sci USA 85:5478-5482. Gingrich JA, Caron MG (1993) Recent advances in the molecular biology of dopamine receptors. Annu Rev Neurosci 16:299-321. Goldstein M, Deutch AY (1992) Dopaminergic mechanisms in the pathogenesis of schizophrenia. FASEB J 6:2413-2421.

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